1. Field of the Invention
The present invention relates to rotavirus antigens of which the mass production via cell culture are difficult, a vaccine against rotavirus infections and a diagnostic agent of the diseases and methods for producing the same. More specifically, the present invention relates to a vaccine and a diagnostic agent useful for the prophylaxis and diagnosis of rotavirus infections, and rotavirus antigens as the effective ingredient thereof. The present invention makes contributions to the prophylaxis and diagnosis of rotavirus infections in humans, in particular.
2. Description of the Related Art
Rotavirus is a pathogenic microorganism causing diarrhea in humans, monkeys, dogs, cats, horses, cows, pigs, sheep, rabbits, rats, chickens, turkeys and the like, and is widely distributed all around the world. Rotavirus infections in humans in particular have been drawing attention, as vomiting and diarrhea in babies, winter-term diarrhea in babies, diarrhea with white feces, kid pseudo-cholera and the like. The prophylaxis and diagnosis thereof have been expected strongly at a worldwide scale.
What will be described below has been known concerning rotavirus.
Morphology and Genome Structure
According to the 6-th report by the International Committee of Taxonomy of Viruses, the virus belongs to the family Reoviridae, and is in an exact icosahedron of a diameter of about 70 nm, comprising a double capsid structure of inner and outer capsids (three layers in total, including the intermediate layer) with no envelop, and has a core of a diameter of about 50 nm at the center of the inner capsid. Inside the core is present a genome, comprising a linear double-stranded RNA with 11 segments, and the sizes of these genome segments are within a range of 0.6 to 3.3 kbp ("Virus Taxonomy: Sixth Report of the International Committee of Taxonomy of Viruses", Archives of Virology, Supplement 10, pp.219-222, 1995).
Genotype and Serotype
From the standpoint of developing a vaccine and a diagnostic agent therefor, attention has been focused particularly on the fourth genome segment and the ninth genome segment (corresponding to the seventh or eighth genome segment in some strain), among the 11 genome segments described above. Rotavirus strains isolated worldwide have been classified into six groups (serogroups) from A to F and each group is divided into various serotypes; for convenience, additionally, rotavirus strains are broadly grouped depending on the genotypes. These isolated strains have a variety of both antigenicities and genotypes ("Fields Virology", 3rd ed., vol.12, pp.1625-1629, edited by B. N. Fields et al., Lippincott-Raven Publishers, 1996, USA). The following description is about what is described immediately above.
The fourth genome segment encodes the structure protein VP4 exposed in a spike form on the surface of the virus particle, and it is reported or assumed that VP4 may functionally be involved in blood cell agglutinins, neutralizing antigens, the infectivity promoted by protease, pathogenicity, membrane fusion, adsorption to cells, and so on. Based on the amino acid sequence of the VP4 and the homology with the RNA or cDNA of the gene, rotavirus stains are currently classified into 20 genotypes (referred to as "P genotype" hereinbelow); and based on the neutralizing test, rotavirus strains are also classified into 10 or 14 serotypes (referred to as "Serotype P" hereinbelow).
The ninth genome segment (seventh or eighth genome segment in some strain) encodes the outer capsid VP7 of the virus particle, and it is reported or assumed that the VP7 may possibly function to retain the epitope of neutralizing antigen and two hydrophobic regions. Based on the neutralizing test of the VP7, rotavirus strains are classified into 14 serotypes (referred to as "serotype G" hereinbelow).
Classification of Rotavirus Strains
Among a great number of various isolated strains reported previously ("Fields Virology", 3rd ed., vol.2, p.1627), a typical human rotavirus strain of the group A and the following strains isolated and reported by the present inventors, namely AU-1 (Journal of Clinical Microbiology, 25, 1159-1164, 1987), AU32 (Microbiologyand Immunology, 34, 77-82, 1990) and AU64 (Archives of Virology, 19, 67-81, 1991), are broadly classified as follows in Table 1.
TABLE 1 ______________________________________ Name of strain Serotypes G and P [genotype] ______________________________________ Wa G1P1A [8] DS-1 G2P1B [4] P G3P1A [8] AU-1 G3P3 [9] Hochi G4P1A [8] ST3 G4P2A [6] AU32 G9P1A [8] AU64 G1P1B [4] ______________________________________
Detection Frequency of Serotype
According to about 20 research reports worldwide about the detection frequency of the serotype G of rotavirus, type G1 is main, occupying about 60 to 85% in Japan, European countries, Australia and Central Africa and the like, while the remaining part is primarily occupied by types G2 and G3. In India, Thailand, Bangladesh, Mexico and the like, alternatively, G1, G2 G3 and G4 are detected in a sporadic fashion, and the ratio of them in total ranges at about 20 to 80%, and the sporadic occurrence of other types except these types is also observed prominently.
Virus Culture
Because rotavirus isolated from monkeys and cattle is generally grown in a culture cell in a relatively ready manner, the culturing or passages thereof is not essentially hard. However, so-called abortive infection occurs during the passage of human rotavirus in cell culture, characteristically involving the generation of a virus antigen impossible of serial passages for obtaining the infectious virus particle, and therefore, the virus passage is very hard. Hence, a procedure has been designed, comprising preparing a reassortant between a human rotavirus with a low growth potency and a monkey- or cow-derived rotavirus with a high growth potency, to improve the growth potency of human rotavirus, or comprising inoculating a human rotavirus preliminarily treated with trypsin into a monkey-derived cell strain M104 (ECACC No. 85102918) and AGMK (African Green Monkey Kidney) cells, and thereafter injecting the rotavirus to the roller tube culture using a maintenance medium supplemented and mixed with trypsin. Currently, almost all human rotaviruses of the group A can directly be cultured or isolated in laboratories. However, even through the above roller tube culture, a virus antigen at an amount required for the production of vaccine or diagnostic agent cannot be recovered. Still currently, the culturing or passaging of rotaviruses except the viruses of the group A is at a very difficult stage.
Host of Virus Culture
For the culturing of permissive cells permitting the proliferation of rotavirus, the following individual cell strains have been known, other than the MA104 and GMK cells; individual cells of FBK (fetal bovine kidney), CMK (cercopithecus monkey kidney), MK (crab-eating macaque kidney), etc.; individual cell strains of monkey-derived CV-1, FRh L2, BSC-1, and Vero cell and dog-derived MDCK, human intestinal epidermis-derived CaCO-2, etc. (WO 92/01784, Japanese Patent Laid-open No. 06-328107, the "Fields Virology", 3rd ed., vol.2, pp.1647-1648, pp. 1661-1162). For the mass production of rotavirus antigen for the purpose of producing a vaccine, furthermore, cell strains for use in producing other virus vaccines, such as Vero, DBS-FLC-1, DBS-FLC-2, DBS-FRh L2, ESK-4, HEL, IMR-90, MRC-5, MRC-9, WI-38, and WRL68, can be utilized ("ATCC Microbes & Cells at Work", 2nd ed., p.144, American Type Culture Collection 1991, USA).
Vaccine
Various vaccines against human rotavirus infections have been attempted and developed since around 1985. The main stream lies in the development of a live vaccine by so-called Jennerian approach, wherein a non-human-derived attenuated vaccine with a similar antigenicity is used as vaccine in place, like small pox vaccine; for example, it has been known clinical trials of live vaccines by using a cow- or monkey-derived attenuated vaccine or an attenuated reassortant between two virus strains each derived from both humans and cow or from both humans and monkeys (WO 92/01784; EPO 130906; Japanese Patent Laid-open No. 06-328107; "Modern Vaccinology", pp. 213-229, edited by E. Kurstak, Plenum Medical Book Company 1994, USA; "Vaccines", 2nd ed., pp. 809-822, edited by S. A. Plotolokin and E. A. Mortimer, W. B. Saundors Company 1994, USA; "The Jordan Report--Accelerated Development of Vaccines 1996", p. 46 and p.68, National Institute of Health issued, USA). As the data of the clinical trials of these live vaccines or the oral dosing thereof has been accumulated, however, it has been remarked increasingly that the prophylactic effect is not sufficient. Currently, therefore, an attempt has been made for the production of a vaccine based on a different idea from the idea of the Jennerian approach.
Diagnostic Agent
Antigens are mainly detected with polyclonal or monoclonal antibodies, and kits for EIA (enzyme immunoassay), ELISA (enzyme-linked immunosorbent assay), latex agglutination and passive hemagglutination, for example, are now commercially available. Diagnostic methods at gene levels by means of PAGE-SS (polyacrylamide gel electrophoresis with silver stain), PCR (polymerase chain reaction) and the like are now developed ("Principles and Practice of Infectious Diseases", 4th ed., vol.2, pp. 1450-1451, edited by G. L. Mandell et al., Churchill Livingstone 1995, USA). Because no diagnostic agent by means of virus antigens for antibody detection has been distributed yet, data relating to the temporal variation of various antibodies against rotavirus antigens in patients is still likely to be insufficient, although such data is extremely useful for the elucidation of rotavirus infections and the countermeasure thereof including prophylaxis and therapeutic treatment.
In such circumstance concerning human rotavirus as has been described above, the present invention has been attained. It is an object of the present invention to overcome the current stage such that the mass production of rotavirus antigen via cell culture is very difficult, and provide a method for producing a rotavirus antigen at a large scale, the antigen being required for producing a vaccine against rotavirus infections, and a diagnostic agent of the diseases.
It is the other object of the present invention to provide the antigen recovered by the method described above, a vaccine against rotavirus infections and a diagnostic agent of the diseases, which are to be produced by using the antigen.